Abstracts: AACR Special Conference: Translation of the Cancer Genome; February 7-9, 2015; San Francisco, CA

Abstract

Next-generation sequencing has yielded unprecedented insights into the mutational landscapes of cancer cells. The next challenge is to decipher the functional relevance of these mutations for cancer proliferation, metastasis and response to therapy. To tackle these questions, we have developed a versatile functional-genomics platform that overcomes issues that have plagued traditional approaches. Most recently, we have established two novel screening technologies based on the bacterial CRISPR/Cas9 system. In our CRISPRi approach, a catalytically dead version of Cas9 (dCas9) fused to a KRAB transcriptional repressor domain is targeted to specific DNA loci close to the transcription start site of endogenous genes to block transcription. Importantly, and in contrast to RNAi-based approaches, CRISPRi is highly specific and virtually lacks off-target effects. This specificity enabled us to construct more condensed genome-wide libraries. Importantly, CRISPRi proved to be non-toxic and reversible. We also developed an approach for targeted activation of endogenous genes, termed CRISPRa, in which dCas9 recruits an array of transcriptional activators to a transcription start site of interest. CRISPRi-based loss-of-function screens and CRISPRa-based gain-of-function screens yield rich, complementary insights into cellular pathways. In particular, drug resistance in cancer cells is commonly driven by gain-of-function events, which we can model using CRISPRa. In a pilot application, we identify genes whose expression levels control growth and survival of human leukemia cells.

While primary genetic screens generate a list of hit genes, it commonly is challenging to elucidate the functional connections between these genes. Systematic, high-density mapping of genetic interactions is a powerful approach to elucidate functional pathways and reveal synthetic lethal / synthetic sick gene pairs, and has successfully been applied in microorganisms. We have developed a functional genomics platform that enables the parallel measurement of 100,000s of double-mutant phenotypes in mammalian cells for the construction of high-density genetic interaction maps. This platform reveals novel biological pathways in cancer cells and points to synthetic-lethal / synthetic-sick gene pairs, which can be exploited therapeutically. It also makes it possible to systematically uncover the accessible escape routes to targeted therapies, paving the way for rational design of combination therapies that pre-empt cancer drug resistance. Systematic comparison of genetic interactions between different growth conditions and genetic backgrounds can reveal context-specific rewiring of functional networks.

We present the application of our platforms to identify adaptations and vulnerabilities in the stress response network of leukemia and multiple myeloma cells. Stress response pathways play important roles in cancer cell survival, drug resistance and tumor progression, and cancer cells are particularly dependent on such pathways. A prominent example of such non-oncogene addiction is the hypersensitivity of multiple myeloma cells to proteasome inhibition. We are applying our functional genomics platforms to understand the rewiring of genetic networks in myeloma cells that underlies this non-oncogene addiction and systematically characterize synthetic-lethal vulnerabilities that are potential new targets for combination therapies.